SRC TR-10-066

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT

Dibromomethane, CAS No. 74-95-3

Genotoxicity – Chromosomal Aberrations

Prepared by:

Ashlee Aldridge, Justine Von Runnen, and Joan D. Garey

Environmental Science Center SRC, Inc.

7502 Round Pond Road North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.0.009.004

Submitted to:

OPPT/RAD/HPVCB (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue

Washington, D.C. 20460

Elizabeth Margosches, Project Officer Meena Sonawane, Work Assignment Manager David Brooks, Alternate Wo rk Assignment Manager

March 5, 2010HPV1 Test Rule – Data Adequacy Assessment

Dibromomethane, CAS No. 74-95-3

Genotoxicity – Chromosomal Aberrations

March 2010

Submitted to: High Production Volume Chemicals Branch

Risk Assessment Division

Office of Pollution Prevention and Toxics Environmental Protection Agency

WAM: Meena Sonawane

Alternate WAM: David Brooks 1200 Pennsylvania Avenue, NW

Washington, DC 20460-0001

Prepared By:

SRC, Inc.

Environmental Science Center 2451 Crystal Drive, Suite 804 Arlington, VA 22202Draft HPV1 Test Rule – Data Adequacy – Chromosomal Aberrations CAS No. 74-95-3

PREPARATION OF THE DAT A ADEQUACY ASSESSMENT

This document contains the review of a toxi city study conducted in response to the first

HPV TSCA Section 4 Test Rule (71 FR 13708, March 16, 2006). Using the applicable test guideline and professional judgment as guide s, the study was reviewed for adequacy and evaluated for compliance with the test guideline. (T he applicable test guid eline is not always the test guideline required by the test rule, because some test sponsors requested and received approval from EPA to use a different guideline .) The study was summarized according to a standard format, assessed for adequacy, and assi gned a Klimisch reliability code. If the study was determined to be inadequate, a basis for th at conclusion is supplie d. For studies already assigned Klimisch codes, an explanation is prov ided if the reviewer’s assessment of data adequacy differed from the pre-assigned Klimisch code.

A detailed evaluation of the test report a nd study conduct is provided at the end of the summary document in a section entitled “Revie w of TSCA Section 4 Final Study Report”. Any deviations from the applicable test guideline are described. The study is also evaluated for compliance with Standard Operating Procedur es, Good Laboratory Prac tice Standards, and Quality Assurance procedures, as well as the use of appropriate controls and test substances. Any deficiencies in the descri ptions of the study method, test system, animals studied, physical conditions, dosage information, indi vidual animal data, calculations, and statistical analyses are described, and internal inconsistencies within the study report are identified. An overall assessment is provided for each of the following: study conduct, data reliability, and data utility.

1Draft HPV1 Test Rule – Data Adequacy – Chromosomal Aberrations CAS No. 74-95-3

GENOTOXICITY – CHRO MOSOMAL ABERRATIONS

Title: Chromosome Aberration Test in Human Lymphocytes In Vitro

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Dibromomethane, CAS No. 74-95-3

Remarks: Clear colorless liquid; Purity: 99.4%; indi vidual samples of dibromomethane were supplied by both Co-Sponsors. These two test mate rials were then mixed in equal proportions and the resulting mixture anal yzed to determine its purity.

METHOD

Method/guideline followed: OECD Guidelines for Testi ng of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 2000/32/EC. The study design also meets the requir ements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity and US OPPTS 870.5375 and 40 CFR 799.9537 guidelines.

Test type: In vitro mammalian chromosomal aberration test

GLP compliant?: Yes

Year study performed: 2006

Test species: Human lymphocytes

Metabolic activation: With and without S9 (from the livers of male Sprague-Dawley rats weighing ~ 250 grams).

Concentrations tested: Preliminary Toxicity Test: 6.80, 13.59, 27.19, 54.38, 108.75, 217.5, 435, 870 and 1740

μ g/mL with and without S9 Experiment 1: 27.19, 54.38, 108.75, 217.5, 435 and 870 μg/mL with and without S9 Metaphase Analysis: 54.38, 108.75 and 217.5 μ g/mL with and without S9

Statistical methods used: The frequency of cells with aber rations excluding gaps and the frequency of polyploid cells we re compared, where necessary, with the concurrent vehicle control value using Fi sher's Exact test.

Test method/conditions remarks: A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and wit hout metabolic activation followed by a 20-hour recovery period, and a continuous exposure of 24 hours without metabolic activation. The dose

2Draft HPV1 Test Rule – Data Adequacy – Chromosomal Aberrations CAS No. 74-95-3

range of test material used was 6.80, 13.59, 27.19, 54.38, 108.75, 217.5, 435, 870 and 1740

μ g/mL. Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test material precipitate ob servations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

Using a qualitative microscopic evaluation of th e microscope slide preparations from Each treatment culture, appropriate dos e levels were selected for mi totic index evaluation. Mitotic

index data was used to estimate test material toxi city and for selection of the dose levels for the main study.

In the main study, duplicate cultures of human lym phocytes, treated with the test material, were

evaluated for chromosome aberrations at three dose levels, together with vehicle and positive

controls. Two treatment conditions were used fo r the study, i.e. In experiment 1, 4 hours in the presence for an induced rat liver homogenate metabolizing system (S9), at a 2% final

concentration with cell harves t after a 20-hour expression peri od and 4 hours exposure in the

absence of metabolic activation (S9) with a 20-hour expression. Experiment 2 was performed but not scored because a clear positive result was obtained in Experiment 1.

The dose levels used in Experiment 1 were select ed on the basis of the results of a Preliminary toxicity test and were 27.19, 54.38, 108.75, 217.5, 435 and 870 μ g/mL for the 4(20)-hour exposures both with and without S9. The dose levels selected for me taphase analysis were 54.38, 108.75 and 217.5 μ g/mL in both the absence and presence of S9. The dose levels used in Experiment 2 were 54.38, 108.75, 217.5, 435, 652 .5 and 870 μ g/mL in the absence of S9, and 13.60, 27.19, 54.38, 108.75, 217.5 and 435 μ g/mL in the presence of S9.

Vehicle (dimethyl sulphoxide, DMSO) and positive cont rols were used in parallel with the test material. The positive control materials we re Mitomycin C (MMC) dissolved in Minimal Essential Medium (0.4 μg/mL) in the absen ce of activation and Cyclophosphamide (CP) dissolved in DMSO (5 μg/mL) in the presence of activation.

A total of 2000 lymphocyte cell nuclei were coun ted and the number of cells in metaphase recorded and expressed as the m itotic index and as a percentage of the vehicle control value.

RESULTS

Genotoxicity result: Positive

Cytotoxic concentration (if applicable): ≥ 870 μg/mL

Results remarks: All vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the freque ncy of cells with aber rations indicating the satisfactory performance of the test and of the activity of the metabolizing system.

The test material induced a statistically significant increase in the frequency of cells with aberrations at all dose levels analyzed, in both exposure groups, using a do se range that included

3Draft HPV1 Test Rule – Data Adequacy – Chromosomal Aberrations CAS No. 74-95-3

a dose level that induced greater than 50% mitotic inhi bition. No increase in the incidence of polyploid cells was observed in either the absence or presence of S9.

The study was terminated after Experiment 1 becau se a statistically significant increase in

structural aberrations were obser ved in the 4(20)-hour exposures for Experiment 1. The response

was considered to be clear evidence of clas togenic activity and in such cases a second experiment is not necessary and not required by the test guidelines.

CONCLUSIONS

The test material induced a statistically significant dose-related increase in the frequency of cells

with chromosome aberrations in both the absenc e and the presence of metabolic activation. The material was considered to be clastogenic to human lymphocytes in vitro

STUDY RELIABILITY

[1] Reliable without restrictions

REFERENCE

EPA-HQ-OPPT-2005-0033-0254.5, Durward et al . (2007) “Dibromomethane: Chromosome Aberration Test in Human Lymphocytes In Vitro ”. SPL Project Number 0466/0262, 1-27 pgs. Sponsor: Bromine Compounds Ltd. and Albemarle Corporation Health, Safety and Environment.

4Review of TSCA Section 4 Final Study Report

CITB Project Manager: Catherine Roman

Chemical Name: Dibromomethane

CAS Registry Number: 74-95-3

Report Title: Chromosome Aberration Test in Human Lymphocytes In Vitro

Test Sponsor: Bromine Compounds Ltd. and Albemarle Corporation

Testing Lab: Safepharm Laboratories Limited

Test Standard: 799.9537

Date Study Received by EPA: June 07, 2007 Date Study Due to EPA: May 17, 2007

Special Instructions (e.g., regarding approved protocol modifications, etc.):

Part I. Test Report and Study Conduct

Enter one of the following codes into each of the checkboxes 1 through 13: Agree [ A ], Disagree [ D ], Not Applicable [ NA ], or Cannot Determine [ C ], and add comment, below, as needed

[D] 1. The final study report indicates evidence of gross, unauthorized or undocumented deviation from the EPA test standard requirements, including “shall,” “must,” or “will” violations. List significant test standard deviations and “shall,” “must,” or “will” violations in comments below.

[D] 2. The final study report indicates evidence of deviations from the “should” statements in the study protocol that may have a bearing on the study conclusions. List these in comments below.

[NA] 3. Approved modifications of test standard or testing protocol (CITB PM describe or attach) were not followed or are inadequately documented.

[D] 4. There is evidence that laboratory Standard Operating Procedures (SOPs) were inadequately followed.

[D] 5. The Statement of Quality Assurance (QA) inspections, their dates, and findings were missing or not signed. There is evidence that QA procedures may be inadequate.

[D] 6. There is evidence that Good Laboratory Practice Standards (GLPS) were inadequately followed.

[D] 7. The test, control, or reference substances are different from that specified in the section 4 test rule or

Enforceable Consent Agreement (ECA).

[D] 8. Animal identification is inadequate or unclear; use of unhealthy animals or other factors (e.g., deviations from temperature, lighting, dosing) are apparent.

[D] 9. The final study report inadequately describes the study method.

[D] 10. The final study report inadequately describes the test system.

[D] 11. The final study report inadequately describes the dosage information.[D] 12. The final study report does not accurately reflect the individual animal data (or that of other discrete observations pertinent to the type of study conducted), or individual data were not provided.

[D] 13. The final study report inadequately describes the calculations, interpolations, data transformations, statistical analyses, or modeling parameters used.

Part II. Conclusions

Place a check mark beside the summar y statement(s) that apply to this review and add comments below as appropriate

Part A. To be filled out by Reviewer

Study Conduct 1. The study was well-conducted. [X] 2. The study was generally well-conducted, with some minor [ ] deficiencies (list in notes below). 3. The study conduct was significantly deficient or questionable, (give [ ] reasons in notes below).

Data Reliability 4. The study results are precise and accurate. [X] 5. Appropriate methods were used to determine [X] statistical significance. 6. If repeated, the test results would be expected to be the [X]

same.

Data Utility 7. The data are useful for ri sk assessment for the endpoint tested. [X] 8. The data are useful for some other purpose (e.g., dose-setting). [ ] 9. The data are not useful for any purpose (describe in notes below). [ ]

Reviewer Comments (continue on separate page, if needed):

Reviewer Name/Title/Bran ch/Division/Office: Ashlee Aldridge/SRC, Inc.

Reviewer Signature: David Brooks/HPVCB/RAD/OPPTS

Part B. To be filled out by CITB PM

Tiered Testing Decision 1. The tiered testing trigger was me t, additional testing is indicated. [ ] (when applicable) 2. The trigger for additional (tiered) testing was not met. [ ]

Study Acceptability 3. The study is acceptable. [ ] 4. The study may not be acceptable [ ] (provide rationale in memo to CITB chief).